Transcript Synapse Complexity Swartz Foundation Banbury Meeting, CSHL 15th April 2009 Seth Grant Genes to Cognition program www.genes2cognition.org Wellcome Trust Sanger Institute, Cambridge UK.

Synapse Complexity
Swartz Foundation
Banbury Meeting, CSHL
15th April 2009
Seth Grant
Genes to Cognition program
www.genes2cognition.org
Wellcome Trust Sanger Institute, Cambridge UK
MASC
Post-Synaptic Proteome
Presynaptic terminal
MASC
Postsynaptic terminal
PSD
MASC MAGUK Associated Signaling Complex
PSD
Post Synaptic Density
-----------------------------------------------------------------------------------------PSP
Post Synaptic Proteome (total set of postsynaptic proteins)
Genetic evidence for postsynaptic complexes
NR2
NMDA receptor
fyn
Grant, et al,
Migaud et al,
Sprengel et al,
MAGUK proteins
PSD-95
Science. 258, 1903-10. 1992
Nature, 396; 433-439. 1998
Cell 92, 279-89. 1998
Learning impairments
Plasticity impairments
Proteomic characterisation of complexes

NR
PSD95
• 2.5 Mda
• 77 proteins
• 186
• 158
• 118 core
Husi et al.
Husi & Grant.
Collins et al,
Fernandez et al,
Nature Neuroscience, 3 (7), 661-669. 2000.
J. Neurochem, 77, 281-291. 2001
J. Neurochem. 2005
submitted
(2000)
(2005)
(2009)
(2009)
NRC / MASC
2-3 MDa
186 proteins
47 genes in human disease
48 synaptic plasticity
42 rodent behaviour
synaptic strength
gene expression
mRNA turnover
Protein turnover
Migaud et al, Nature, 396; 433-439. 1998;
Husi et al, Nature Neuroscience, 2000
Behaviours
Cognition & plasticity
Complexity
PSD
700 -1500 proteins
MASC
MASC
77 - 185 proteins
MASC occupies ~10 % of the PSP
- a core subset
- same classes of proteins
- ideal model of PSP
- multiple isolation methods available
PSD
Post Synaptic Density
ER:microsomes
Splicesome
NRC/MASC
Nucleolus
Peroxisomes
Mitochondria
Phagosomes
Golgi
Choroplasts
Lysosomes
Exosomes
Grant. (2006) Biochemical Society Transactions. 34, 59-63. 2006
1124
491
311
186
147
181
179
140
81
81
27
21
Is there some logic to this complexity ?
Molecular Networks: modular protein interaction networks
Pocklington, et al, Molecular Systems Biol 2006.
Upstream and Downstream modules
upstream
downstream
A useful tool for handling complexity
Human cortex PSD: 1462 protein network
Alex Bayes, Mark Collins, Louie Van De Lagemaat,
Ian Whittle, Jyoti Choudhary
The origin and evolution of synapses and the brain
Emes et al, Nature Neuroscience 11, 799 (2008)
Tree of life
6 myr
75 myr
600 myr
1000 myr
eukaryotes
metazoans
chordates
6 myr
75 myr
600 myr
Origin of the brain
1000 myr
The first neurons and first brains
combinations
expansion
protosynapse
Emes et al, Nature Neuroscience 11, 799 (2008)
Comparative genomics
570 genes: 186 NRC/MASC; 570 PSD
Number of orthologues compared to mouse
Synapse orthologues in single cell eukaryotes control
response to environment
143 PSD proteins
Yeast behaviours:
-Environmental responses
-- stress
-- nutrients
-- pH
Origin of the brain
neuron first model
synapse first model
eukaryotes
metazoans
chordates
6 myr
75 myr
600 myr
Origin of the brain
1000 myr
protosynapse
Evolutionary elaboration and expansion of the protosynapse
Unicellular
eukaryotes
Invertebrates
Vertebrates
upstream
downstream
Origins of functional classes
upstream
Method: Proportion of each functional class with
earliest identifiable orthologue
downstream
Y
I
V
yeast
invertebrate
vertebrates
Evolution of ‘learning molecules’
• neurotransmitter receptors
• second messengers
• protein synthesis
GO terms
Y
W
F
Z
C
M
H
yeast
worm
fly
zebrafish
chicken
mouse
human
Increased combinations of complexes in vertebrates
from expansion in paralogues
NRC / MASC
Invertebrates
Vertebrates
NR2
NR1
Proteins
DLG
NR1
NR2
DLG
1
1
1
Complexes
1
Theoretically possible MASC combinations in mammals
1
4
4
16
>1030
Synapse evolution
Protosynapse
‘first synapse’
Synapse evolution
Protosynapse
‘first synapse’
first neurons
Synapse evolution
Protosynapse
‘first synapse’
first neurons
Brain size vs Synapse complexity
Big synapse proteomes evolved before big brains
6 myr
75 myr
600 myr
1000 myr
How are complex synapses used in complex brains ?
Examine synapse proteome composition in different in classes of neurons in mouse
Chris Anderson, Cathy Vickers, Andrew Pocklington
anatomical expression level profiling
>150 MASC/PSD proteins in 22 regions of mouse brain
Measured:
mRNA
• in situ
• microarray
protein
• immunohistochem
• western
variation in levels & overlap
Combinations of synapse proteome define brain regions, neuron types
MASC
Expression
barcode
anatomical
variation
phylogeny
recent
upstream
0 4 4 3 1
1 4 3 2 3
downstream
2 3 2 2 1
ancient
MASC
Expression
barcode
anatomical
variation
phylogeny
recent
upstream
0 4 4 3 1
1 4 3 2 3
downstream
2 3 2 2 1
ancient
Protosynapse has most conserved & uniform expression pattern
Evolutionary expansions gave combinations used to generate anatomical diversity
Combinations
Signaling networks in the PSP
a complex combinatorial signalling machine
NMDA receptor activation with a synaptic plasticity protocol
how many substrates change?
NMDA stimulation
(long term depression, LTD)
> 200 phosphorylation sites.
> 120 proteins
PSD
Marcelo Coba, Andrew Pocklington, Mark Collins, Jyoti Choudhary
(Science Signalling 2009)
NMDA receptor activation with a synaptic plasticity protocol
how many kinases change?
9 of 21 kinases tested
Marcelo Coba
Combinatorial outputs
10 phosphorylation sites on 4 proteins
Stimulus:
No change
Increase phos
Decrease phos
Marcelo Coba
Combinatorial outputs
10 phosphorylation sites on 4 proteins
Stimulus:
No change
Increase phos
Decrease phos
Combinatorial outputs
10 phosphorylation sites on 4 proteins
Stimulus:
No change
Increase phos
Decrease phos
Combinatorial outputs
10 phosphorylation sites on 4 proteins
Stimulus:
No change
Increase phos
Decrease phos
Combinatorial outputs = states
sites
states
GluR1
3
8 (23)
10 proteins
100
1030 (2100)
PSP
104
1060
Are any two synapses the same?
Can a synapse ever be in the same state twice?
What are the physiological constraints?
Postsynaptic kinase-substrate network - mapping 743 phosphorylation events
kinases
substrates
Marcelo Coba, Andrew Pocklington
Building the network
defining minimal units
How many substrates for a kinase?
k1
kinase
divergence
P
P
P
20.5 ( 8.3) protein substrates phosphorylated by each kinase
Number of kinases with n substrates
9
number of kinases
8
7
6
5
4
3
2
1
0
0-5
5-10
10-15
15-20
20-25
n substrate
25-30
30-35
35-40
How many sites were phosphorylated by more than one kinase?
k1
kinase
convergence
k2
kn
• redundance / robustness
• multiple upstream receptors
P
65% (129) sites phosphorylated by multiple kinases
Number of sit es wit h n kinases
80
number of sites
70
60
50
40
30
20
10
0
1
2
3
4
5
n kinase
6
8
9
10
12
Hubs – highly connected
Proteins
Sites
coincidence detectors
NMDA
receptor
mGluR
receptor
Dopamine
receptor
Combinations of sites
Functional orchestration
Synapse evolution and expansion model of synapse diversity and behaviour
2
5
0
2
0
0
BEHAVIOUR
EPM
1
5
0
1
0
0
5
0
2
0
0
2
0
4
0
6
0
OF
NOE
FC
RR
Acknowledgements
Proteomics
Marcelo Coba
Alex Bayes
Support
Bilal Malik
Mark Collins
Jyoti Choudhary
Expression
Christopher Anderson
Cathy Vickers
Informatics
Andrew Pocklington
J. Douglas Armstrong
Mike Croning
Richard Emes
www.genes2cognition.org
www.g2conline.org
Wellcome Trust
Medical Research Council
European Union